Abstract

Blastogenesis, the classical assay of lymphocyte function, measures the ability of lymphocytes to incorporate thymidine, one of the basic building blocks of DNA. This assay has long been used as an indicator of severe immunodeficiency when mitogens (non-specific stimulators) incubated with cells at optimal concentrations fail to activate them. Recently, suboptimal mitogen concentrations have been used to aid in measuring stress and its effects on the immune system. Due to the universal nature of nucleic acids, this assay can be used with any species. Therefore, it was chosen as a method for evaluating immune health of captive killer whales in an ongoing study. Peripheral blood samples were analyzed at the originating park for CBC and blood chemistries on a routine basis (every 2 to 4 weeks). Parallel blood samples for lymphocyte function were obtained every two months and transported to the Davis laboratory via next-day delivery service. Animals with apparent clinical or behavioral problems were sampled more often. Isolated mononuclear leukocytes were incubated with either ConcavalinA (ConA) or Phytohemagglutinin (PHA) at optimal and suboptimal concentrations (1 and 0.1 μg/ml, respectively). Cells were incubated with mitogen for 72 hours before either [³H]-thymidine or 5-bromo-2'-deoxyuridine (BrdU) was added. After an additional incubation of 18 hours, cells were analyzed for the presence of the thymidine analog. High levels of incorporation represent significant cell activation and DNA synthesis.

Most animals in this study performed well at optimal mitogen concentrations; this was to be expected as no life-threatening chronic clinical diseases suggestive of severe immunodeficiency were noted during the study. However, variations in suboptimal mitogen-induced blastogenesis responses were noted both between animals and within individual animals over time. Establishing a numeric ratio of optimal to suboptimal response best identified these fluctuations. Animals exhibiting acute and or chronic infectious disease processes typically presented with increased ratios of blastogenesis due to a decrease in the response to suboptimal mitogen. In addition, many animals experiencing environmentally induced stress demonstrated similar changes. This data suggests that employing blastogenesis assays with optimal:suboptimal mitogen ratios can be an important tool in managing the health of captive marine mammals.